Fusion proteins are proteins created through the joining of two or more genes which originally code for separate proteins. Translation of this fusion gene results in a single polypeptide with functional properties derived from each of the original proteins. Recombinant fusion proteins are created artificially by recombinant DNA technology for use in biological research or therapeutics.
Several techniques are available for producing fusion proteins which retain the desirable characteristics of thermostability, solubility and a high level of expression.
The most commonly used method for producing fusion proteins is use of fusion tags. Examples of popular fusion tags include, Histidine-tag, glutathione-s-transferase (GST), Maltose binding protein, NusA, thioredoxin (TRX), polyhistidine (HIS), small ubiquitin-like modifier (SUMO) and ubiquitin (Ub).
One of the strategies provides a method to express protein of interest as a staphylokinase (SAK) fusion. Since one can easily assay SAK activity using the simple chromogenic assay, one could adopt the SAK assay as a measure of successful refolding of the SAK fusion protein. SAK is a 136 amino acid long bacteriophage encoded protein of 15.5-kDa size and is devoid of disulphide linkages. SAK is presently undergoing clinical trials for blood clot-lysis in the treatment of thrombovascular disorders due to its ability to convert plasminogen, (an inactive proenzyme of the fibrinolytic system) into plasmin, which is a protease. SAK has gained importance as a potential therapeutic thrombolytic protein and is an extracellular protein produced by Staphylococcus aureus strains. It is also produced by S. lyicus, S. simulans, S. seweri and S. xylosus. Schlott et al 273(35): 22346-50, 1998, have disclosed that SAK is not an enzyme, but rather a cofactor; it forms a 1:1 stoichiometric complex with plasmin-(ogen) that converts other plasminogen molecules to plasmin, a potent enzyme that degrades proteins of the extracellular matrix. The high affinity of the SAK-plasminogen complex for fibrin makes it a promising thrombolytic agent.
Jackson and Tang, Biochemistry, 21(26): 6620-5, 1982 have reported that SAK has been shown to be homologous to serine proteases although it does not have any protease activity of its own. Sakharav et al J. Biol. Chem. 271: 27912-27918, 1996, have reported that SAK structurally resembles plasminogen activators, has plasminogen-binding site and serine protease domain but does not show protease activity.
IN/1813/KOL/2008 describes that SAK has proteolytic activity, Salunkhe et al 1(1): 5-10, 2009, have reported that the expression levels of SAK-IFN were found to be two folds higher than that observed with untagged IFN under similar experimental conditions. It has been observed that a full length SAK (FL-SAK) when expressed results into 2 fragments as mature SAK and signal peptide. It was found that when FL-SAK was expressed in BL21-A1 cells resulted into 2 fragments as mature SAK and signal protein. This proves that SAK has autoproteolytic property when used as C-terminus fusion. Thus, SAK can be used as a proteolytic tool by exploring its autoproteolytic activity. One of the problems associated with use of SAK for the expression of protein of interest is that the protein of interest is also degraded after release of SAK protein from fusion protein.